Affinity chromatography has gained widespread acceptance as a powerful purification tool in the biological sciences. However, certain cases have arisen in which secondary binding interactions appear to dominate. These observations cast doubt on the concept of affinity chromatography. This proposal presents procedures that permit the determination of the mechanisms of binding. Thus, true cases of biospecific chromatography can be identified. Futhermore, these procedures permit the measurement of the strengths of interaction of proteins with immobilized ligands as well as soluble ligands. This yields an alternative method for carrying out binding studies. This will be especially valuable for modified or inactive proteins. This project proposes to apply these procedures to a series of classical examples of the affinity method to either verify their validity or expose their shortcomings. It will also extend this approach to new systems.